Skeletal mineralization is fundamentally important to all vertebrate species. Too little mineralization results in structurally-compromised bone that is prone to failure. On the other hand, pain and disability occur when there is inappropriate or ectopic mineralization and calcification of soft tissues. The major mechanisms controlling mineralization are poorly understood resulting in a major gap in knowledge. Our ongoing studies on the genetic basis of opsismodysplasia (OPS), a rare chondrodysplasia that is characterized by a marked delay in endochondral ossification, identified a new potential regulator of matrix mineralization: SH2 Domain-containing Inositol 5-phosphatase 2 (SHIP2). SHIP2 functions as a phosphatase that dephosphorylates phosphatidylinositol (3,4,5)P3 (PIP3) to generate phosphatidylinositol (3,4)P2 (PIP2). Data from our in vitro SHIP2 inhibitor and SHIP2-deletion studies confirmed that SHIP2 deficiency leads to a mineralization defect. Furthermore, experiments on matrix vesicles (MVs) isolated from chondrocytes and osteoblasts demonstrated that the loss of SHIP2 leads to a failure of MVs to support mineral deposition. Together, our data support the overall hypothesis that SHIP2 regulates MV function. SPECIFIC AIM 1: To investigate the regulation of cell surface phosphoinositides (PIs) by SHIP2 in mineralizing cells. Hypothesis 1 states that SHIP2 controls MV formation by regulating PI cell surface levels. We have generated new tools to address this hypothesis including ATDC5 chondrocyte and SaOs-2 osteoblast cell lines engineered using CRISPR to eliminate SHIP2 protein, and several highly specific, fluorescently-labeled PI-binding proteins. These reagents will be used to track the sub-cellular and cell surface PIP3, and metabolites of PIP3 in the presence or absence of SHIP2. SPECIFIC AIM 2: Investigate the control of MV composition by SHIP2. Hypothesis 2 states that SHIP2 regulates mineralization by controlling MV composition. This hypothesis will be addressed in two Sub-Aims: Sub-Aim 2.1: Define the proteins recruited to the cell surface by PIs: PI pull-down experiments will be conducted separately on cell lysate, MV, and membrane preparations using reagents against biosensor proteins. We will focus on PIP3 initially and then other PIPs that have disrupted distribution in the absence of SHIP2. Immunoprecipitated proteins will be identified by mass spectroscopy and confirmed by immunoblotting. Sub-Aim 2.2: Define how SHIP2 controls MV composition: A comprehensive proteomic analyses will be conducted on MV fractions from both skeletal cell types. Mass spectroscopic findings will be confirmed in immunohistochemical and immunoblot experiments. The proposed studies have the potential for new insight into the mechanisms of the fundamental, but poorly understood, process of matrix mineralization. Significantly, these studies may identify new, rational targets for the clinical treatment of bone mineralization defects and other mineralization-associated disorders.